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Abstract:

A drug composition for treating type 2 diabetes and diabetic chronicity
complications is disclosed. The composition is composed of mangiferin and
berberine in a certain ratio. The composition has a preferable
hypoglycemic and hypolipidemic effect compared with mangiferin and
berberine which is used alone

Claims:

1. A composition for treating type 2 diabetes, the composition comprising
mangiferin and berberine, wherein the weight ratio range of mangiferin
and berberine is 1:0.1 to 1:20.

2. The composition according to claim 1, wherein: the weight ratio range
of mangiferin and berberine is 1:1 to 1:10.

3. The composition according to claim 2, wherein: the weight ratio range
of mangiferin and berberine is 1:3 to 1:6.

4. The composition according to claim 1, wherein: berberine is berberine
hydrochloride, berberine sulfate, tannin berberine or a medically
acceptable salt of berberine.

5. A treatment of type 2 diabetes using the composition as claimed in
claim 1.

6. The treatment according to claim 5, wherein the treatment is a
hypoglycemic agent.

7. The treatment according to claim 5, wherein the treatment can prevent
and treat the diabetic chronic complications.

9. A drug for type 2 diabetes, wherein the drug comprises an effective
amount of the composition as claimed in claim 1, and a pharmaceutically
acceptable auxiliary material.

10. The drug according to claim 9, wherein the drug is an oral formulation
in the form of a tablet, a capsule, a gentle capsule, a granule, a pill,
a syrup, an oral solution, or an oral suspension.

Description:

CROSS REFERENCE TO RELATED APPLICATION

[0001]This application is a continuation application filed under 35 U.S.C.
§ 111(a), claiming the benefit under 35 U.S.C. §120 and
§365(c) of a PCT International Application Number PCT/CN2007/002684,
filed Sep. 11, 2007, it being further noted that foreign priority benefit
is based upon Chinese Patent Application 200610122125.7, filed Sep. 12,
2006 in the State Intellectual Property Office of P.R. China, the
disclosures of which are thereby incorporated by reference.

FIELD OF THE INVENTION

[0002]This invention relates to a drug composition for treating type 2
diabetes and diabetic chronic complications, the composition is composed
of mangiferin and berberine.

BACKGROUND OF THE INVENTION

[0003]In recent years, type 2 diabetes and its chronic complications have
become a threat to human health as the "third killer". According to
statistics, standardized prevalence rate of type 2 diabetes in China has
rapidly increased from 0.9 percent in the early 1980's to the current
5.21 percent; and the standardized prevalence rate of impaired glucose
tolerance is 4.76 percent. Long-term hyperglycemia associated with type 2
diabetes results in the damage of many tissues and organs, which in turn
lead to a variety of diabetic chronic complications, such as coronary
heart disease, atherosclerosis, cerebrovascular disease and other
diabetic macrovascular diseases, diabetic nephropathy, diabetic
retinopathy and other diabetic microangiopathy, diabetic neuropathy,
diabetic foot, diabetic maculopathy, diabetic cataract, diabetic
glaucoma, refractive changes, iris and ciliary body diseases.

[0004]At present, most of the oral hypoglycemic agents that are on the
market are expensive or have some adverse reactions, which cause poor
compliance of patients. The pathogenesis of the diabetic chronic
complications is not entirely clear, and there is no ideal drug in the
clinical treatment. Therefore research and development of drugs that are
low cost, but show high efficiency and low toxicity that not only has
hypoglycemic effect but also prevent and treat diabetic chronic
complications will have important clinical significance and market value.

[0005]Mangiferin, a natural polyphenol is from Liliaceae plants such as
Anemarrhena asphodeloides Bunge. etc., molecular weight: 422, structural
formula: C19H18O11, and its chemical structure is as
follows:

[0007]In recent years, more evidence show that the hyperglycemia,
oxidative stress and diabetic chronic complications are closely related.
Muruganandan's research shows that in the streptozotocin-induced diabetic
rats, mangiferin by intraperitoneal injection can control the lipid
peroxidation, and can reduce the glycosylated hemoglobin and serum
creatine phosphate Kinase (CPK) of the diabetes rats and its
complications due to chronic oxidative damage. Mangiferin can also
protect animals avoiding the damage of heart and kidney. [Muruganandan S,
Gupta S, Kataria M, et al. Mangiferin protects the streptozptocin-induced
oxidative damage to cardiac and renal tissues in rats. Toxicology, 2002,
176(3):165-173]. The LD50 of mangiferin by intraperitoneal injection
once is 365 mg/kg in rat [Yu shengmin, Zhong ming. The research advance
of pharmacology effects of the mangiferin. Chinese Journal of Traditional
Medical Science and Technology, 1999, 6(3):199-200]. We can see that
mangiferin is safe.

[0008]Because mangiferin cannot dissolve in water, there is limitation
such as its bioavailability, and formulation.

[0009]Berberine (Ber) is an isoquinoline alkaloid that is extracted from
the root and bark of Coptis chinesis Franch (Ranunculaceae coptis plant).
Berberine is the main ingredient of the coptis. Molecular formula:
C20H18NO4, molecular weight: 336.37. Its chemical
structure is as follows

##STR00002##

[0010]Pharmacological research of berberine shows that it has a variety of
biological activities, such as anti-bacterial, anti-virus, hypoglycemic,
anti-abnormal heart rate, lower blood pressure, anti-tumor,
lipid-lowering, etc.

[0011]Animal experiments show that berberine can reduce the blood glucose
of the normal mice, and the alloxan diabetic and chronic complications
mice, and the spontaneous diabetic and chronic complications KK mice. The
effect is significantly, the duration is long, while berberine can
improve glucose tolerance of the KK mice. In recent years, clinical
research has proven that berberine has the effects of anti-diabetes and
improves its chronic complications, especially in the treatment of type 2
diabetes and chronic complications. The mechanism of the hypoglycemic
effect may be by inhibiting the liver gluconeogenesis and/or promoting
the glycolysis of the peripheral organizations, that is the
non-insulin-dependent. But only under the conditions of moderately high
glucose level (for example, 11.1 mmol/L), will berberine have significant
hypoglycemic effect. However, under conditions of seriously high glucose
level (for example, 22.2 mmol/L), the hypoglycemic effect of the
berberine disappear. Berberine mainly applies to impaired glucose
tolerance, abnormal fasting blood glucose and stable blood glucose of the
diabetics [Yin jun, Hu renming, Chen mingdao, et al. Comparison of
glucose consumption effects of Mmetformin, troglitazone and berberine on
the HepG2 cells. Chinese Journal of Endocrinology and Metabolism, 2002,
18(6):488-489].

[0012]In recent years, studies have shown that sustained hyperglycemia can
increase the activity of aldose reductase in cells which leads to
diabetic neuropathy, diabetic retinopathy and a variety of vascular
diseases. It is significant to inhibit aldose reductase activity on the
treatment of chronic diabetic complications. Many scholars' studies
showed that berberine can inhibit the activity of aldose reductase [Liu
Changshan; Dong Yanhu; Pang Linan; et al. The effects of baicalin and
berberine on proteinuria and glomerular ultrastructure in alloxan-induced
diabetic rats. CHINESE JOURNAL OF DIABETES, 1996, 4(3):163].

[0013]Regarding the treatment dose, berberine is safe. Its side effects
are few. We do not find any adverse reactions when berberine was used for
long. we don't find any adverse reactions when berberine was taken 2.0 g
once orally [Ji yubin. The effective composition and pharmacological
action of Chinese medicine, Harbin: Heilongjiang Science and Technology
Press, 2004, 77].

[0014]The bioavailability of berberine is very low. In a certain blood
glucose range, berberine may play hypoglycemic role. As a result, the use
of berberine in diabetic patients is limited significantly.

SUMMARY OF THE INVENTION

[0015]In order to develop low cost, highly effective and low toxic drug
that not only have hypoglycemic effect but can also prevent and treat
diabetic chronic complications. After a large number of studies, the
inventor finds that the composition that is composed of mangiferin and
berberine in a certain ratio have unexpected effect as follows:

[0016]First, the effect of a composition that is composed of mangiferin
and berberine in the treatment of type 2 diabetes is significantly
enhanced. Therefore, there are obvious synergies between mangiferin and
berberine. It is as follows:

[0017]1. The composition has more prominent hypoglycemic effect, because
non-insulin-dependent hypoglycemic effect of berberine combines with the
insulin-sensitizing effect of mangiferin.

[0018]2, The composition is more effective to prevent and treat type 2
diabetic chronic complications, because the effect of antioxidant and
lipid-lowering of the mangiferin combines with the effect of
lipid-lowering and aldose reductase inhibition of the berberine.

[0019]Second, while mangiferin cannot dissolve in water alone, the
dissolution of mangiferin is increased significantly in the composition
of mangiferin and berberine.

[0020]Finally, the bioavailability of the mangiferin and berberine, when
given alone, is low, but the bioavailability of mangiferin and berberine,
when given as a composition, is improved significantly.

[0021]According to these beneficial effects of the composition, and high
security of berberine and mangiferin single oral administration and low
cost of raw materials of berberine and mangiferin, we can see the
composition will be a low cost, highly effective, low toxic drug, which
the drug not only has the hypoglycemic effect, but also prevents and
treats diabetic chronic complications.

[0022]One aspect of the present invention is to provide a composition that
can be prepared as a treatment of type 2 diabetes, having the following
characteristics: the active ingredient of the composition is composed of
mangiferin and berberine, the weight ratio range of mangiferin and
berberine is 1:0.1 to 1:20.

[0023]The preferred weigh ratio range of mangiferin and berberine is 1:1
to 1:10.

[0024]The more preferred weigh ratio range of mangiferin and berberine is
1:3 to 1:6.

[0030]Yet another aspect of the present invention is to provide a drug,
such that the drug is composed of the composition as defined above with a
pharmaceutical acceptable auxiliary material. The drug may be in the form
of oral formulations, such as tablets, capsules, gentle capsules,
granules, pills, syrup, oral solution, oral suspension, etc.

[0031]Furthermore, another aspect of the present invention is to provide
an effective dose range of the composition as defined above. For in
stance, in rats, the effective dose range is about 20 to 350 mg/day.
Based on the effective dose ranges of different types of animals,
conversion formula imply that the effective dose range will be about 200
to 3500 mg/day/person by oral administration three times or four times
per day. Because of the difference between animals and the human body,
the adjustments of the dose and times in actual clinical application can
be allowed.

[0032]Embodiments of the invention may be explained in detail in the
following examples given below. These examples are provided as further
illustrations of the invention, and therefore should not be construed to
limit the scope of the invention.

EXAMPLES

[0033]Berberine, berberine hydrochloride, berberine sulfate, tannin
berberine, and mangiferin in the invention can be purchased from market
sources, for example at the price of about RMB 1000 yuan/kg. They can
also be obtained by conventional extraction methods, such as those
described here.

Example 1

Preparation of Berberine

[0034]Berberine hydrochloride (more than 98% purity) is dissolved in hot
water. After the pH of the solution is adjusted to 10-12 with ammonia,
the reaction solution is set aside for more than 12 hours. Then the
reaction solution is filtrated, and the deposition is dried, the
deposition is berberine. The purity of berberine is determined to be
98.5% by HPLC.

Example 2

Preparation of Mangiferin

[0035]Rhizoma anemarrhenae is extracted with 75% aqueous ethanol for one
hour twice. The extracts are concentrated. The evaporated extract is
adsorbed by macroporous resin, and then the macroporous resin is eluted
with water adequately. Then the macroporous resin is eluted with 20%
aqueous ethanol, the solution is concentrated to obtain crude
nemangiferin. Then the macroporous resin is eluted with 40% aqueous
ethanol, the solution is concentrated to obtain crude mangiferin. The
crude mangiferin and nemangiferin are recrystallized. The pure mangiferin
and nemangiferin are obtained, their purity is determined to be more than
95% by HPLC.

[0037]Mangiferin, starch and microcrystalline cellulose are mixed
uniformly. An appropriate bond is added into the drug mixture in order to
crank out granule. The granule is dried and then pressed by tablet press
machine. 1000 tablets are prepared. The tablets are coated with a film.
There are 100 mg mangiferin in every tablet.

[0039]Mangiferin and berberine are mixed uniformly, then the drug mixture,
carboxymethyl cellulose and pregelatinized starch are mixed uniformly. An
appropriate bond is added into the drug mixture in order to crank out
granule. The granule is dried and then pressed by tablet press machine.
1000 tablets are prepared. The tablets are coated with a film.

[0041]Mangiferin and berberine are mixed uniformly, then the drug mixture,
microcrystalline cellulose and pregelatinized starch are mixed uniformly.
An appropriate bond is added into the drug mixture in order to crank out
granule. The granule is dried and then pressed by tablet press machine.
1000 tablets are prepared. The tablets are coated with a film.

[0043]Mangiferin and berberine are mixed uniformly, then the drug mixture,
microcrystalline cellulose and pregelatinized starch are mixed uniformly.
An appropriate bond is added into the drug mixture in order to crank out
granule. The granule is dried and then pressed by tablet press machine.
1000 tablets are prepared. The tablets are coated with a film.

[0045]Mangiferin and berberine are mixed uniformly, then the drug mixture,
microcrystalline cellulose and pregelatinized starch are mixed uniformly.
An appropriate bond is added into the drug mixture in order to crank out
granule. The granule is dried and then pressed by tablet press machine.
1000 tablets are prepared. The tablets are coated with a film.

[0047]Mangiferin and berberine are mixed uniformly, then the drug mixture,
microcrystalline cellulose and starch are mixed uniformly. Appropriate
bond is added into the drug mixture in order to crank out granule. The
granule is dried and then pressed by tablet press machine. 1000 tablets
are prepared. The tablets are coated with a film.

[0049]Mangiferin and berberine are mixed uniformly, then the drug mixture,
microcrystalline cellulose and pregelatinized starch are mixed uniformly.
Appropriate bond is added into the drug mixture in order to crank out
granule. The granule is dried and then pressed by tablet press machine.
1000 tablets are prepared. The tablets are coated with a film.

Example 10

Preparation of Berberine Tablets

[0050]The formulation containing: berberine 100 g, and an appropriate
starch is used to prepare 1000 tablets.

[0051]Berberine and starch are mixed uniformly. An appropriate bond is
added into the drug mixture in order to crank out granule. The granule is
dried and then pressed by tablet press machine. 1000 tablets are
prepared. The tablets are coated with a film. There is 100 mg berberine
in every tablet.

[0053]Mangiferin and berberine hydrochloride are mixed uniformly, then the
drug mixture, methylcellulose and pregelatinized starch are mixed
uniformly. An appropriate bond is added into the drug mixture in order to
crank out granule. The granule is dried and then pressed by tablet press
machine. 1000 tablets are prepared. The tablets are coated with a film.

[0055]Mangiferin and berberine tannate are mixed uniformly, then the drug
mixture, sucrose and carboxy methyl cellulose are mixed uniformly. An
appropriate bond is added into the drug mixture in order to crank out
granule. The granules is dried and packed.

[0057]Mangiferin and berberine hydrochloride are mixed uniformly, then the
drug mixture, microcrystalline cellulose and pregelatinized starch are
mixed uniformly. An appropriate bond is added into the drug mixture in
order to crank out granule. The granule is dried and then capsules are
filled 0.1000 capsules are prepared.

[0059]Mangiferin and berberine are mixed uniformly, the macrogol 4000 is
smelt in hot water. Then the drug mixture is mixed uniformly at
75° C., the drug suspension is transferred into storage cans of
pill machine at 75° C.

[0061]The residue refrigerant on pills surface is removed, then the pills
are packed.

Dissolution Rate of the Composition of Mangiferin and Berberine

[0062]Six tablets samples were taken from each of example 3, example, 6
and example, 10. The tablet samples of examples 3 were designated as MAG,
the tablet samples of examples 6 were designated as MB, the tablet
samples of examples 10 are designated as BER. The experiment was
performed in rotating basket method according to the addenda of China
Pharmacopoeia. The samples were put into phosphate buffer solution (PBS,
900 ml, pH 6.8). The rotating speed was 100 r/min. 2 ml solution of
samples were taken out separately at 5 min, 10 min, 20 min, 30 min, 60
min, 120 min, 240 min, 360 min, and 480 min. The same volume of PBS was
supplied at once. The samples solutions were filtrated with microporous
filtration membrane which specification is 0.45 μm.

[0063]The concentration of mangiferin and berberine were determined, the
dissolution rate was accounted. The result is as table 1.

[0066]Berberine is suspended in 1% carboxymethyl cellulose sodium
solution, the concentration of berberine is 25 mg/mL (Sample B). The
composition which is composed of mangiferin and berberine in the ratio of
1:4 is suspended in 1% carboxymethyl cellulose sodium solution, the
concentration of the composition is 50 mg/mL (Sample C).

[0068]Serum samples are taken accurately 100 μL into centrifuge tubes,
then 400 μL acetonitrile-acetic acid (9:1) is added into centrifuge
tubes. The samples are whorled for 1.5 minutes, and then the samples are
centrifuged 3000 r/min for 10 minutes. The supernatant was put into clear
tube, dried by nitrogen, 100 μL mobile phase were put into the
remnants, whorled for 1.5 minutes, then centrifuged 3000 r/min for 10
minutes. The Supernatant was put into sample bottle. All the samples are
determined by HPLC. Injection volume is 20 μL.

[0076]According to the data (Table 2), the pharmacokinetics data of the
composition that is composed of mangiferin and berberine is better than
mangiferin or berberine orally alone. The Cmax, and AUC of
mangiferin and berberine of the composition is better than mangiferin or
berberine orally alone. It suggests that there is interaction in
promoting absorption between mangiferin and berberine.

[0077]In the following experiments, kidney and lens are the representative
organs of diabetic chronic complications.

[0078]The GK (Goto-Kakizaki) rat is the spontaneous model of diabetes, the
pathogenesis of GK rat is very similar to the pathogenesis of type 2
diabetes. After diabetes attack, hyperglycemia and insulin secretion
weakened emerge rapidly, the latter diseases may be complicated by
retinopathy, micro-vascular disease, neuropathy, nephropathy in GK rat.
The GK rats have been widely used in type 2 diabetes study, so we choose
GK rat as the animal model of the following experiment.

The Effect of the Mangiferin, Berberine and Compositions in GK Rats

1. Materials

[0079]1.1 Mangiferin and berberine are prepared in accordance with the
method of preparation of example 1 and 2. Then they can be mixed or
diluted in accordance with the dose and the ratio.

[0082]In addition to the normal rats, the other rats were fed with high
fat diet (20% protein and 3.5% cholesterol).

2. Experimental Group:

[0083]After the rats was feeding adaptation for one week, The GK rats
which the blood glucose level was more than 11.1 mmol/L were selected,
and then divided into the following groups random:

[0084]GK rats control group (n=10); mangiferin groups (10, 30, 200 mg/kg,
each group 10 rats); berberine groups (10, 20, 150 mg/kg, each group 10
rats); composition group 1[[mangiferin (10 mg/kg)+berberine (10 mg/kg)],
10 rats]; composition group 2[[mangiferin (200 mg/kg)+berberine (150
mg/kg)], 10 rats]. Wistar rats are normal control group (10 rats). GK
rats control group and Wistar rats group were given saline. The other
groups were given test drugs. Test samples were administrated orally at
the same time every day for 12 weeks.

3. Measurements:

[0085]3.1 Blood Glucose and lipid: The blood specimens were taken at the
end of the test. Plasma glucose (GLU), triglycerides (TG), total
cholesterol (TC) were determined by HITACHI7080 automatic biochemistry
analyzer.

[0086]3.2 The activity of aldose reductase (AR): At the End of the
experiment, the left side lens of rats was taken, then these lenses were
added into glycerol and put into refrigerator (-80°
C.quadrature) to preserve to determine AR activity. The determination
method of AR activity refer to Maragoudakis's method and change slightly,
that is: the activity of enzyme is indicated by the record of the
absorbance of NADPH in 340 nm decline IOU. This study used two reaction
systems, the first reaction system includes Na+--K+ Phosphate
Buffered Saline (67 mmol/L, pH6.2), DL-glyceraldehyde (10 mmol/L),
2-mercaptoethanol (5 mmol/L), L), NADPH (0.1 mmol/L), and appropriate
quantity enzyme; experimental cup includes all reagents, in the control
cup double distilled water take the place of DL-glyceraldehyde. The
second reaction system is 0.1 mmol/L NADH instead of NADPH, the rest of
the reagent is same as one of the first response system. The activity of
enzyme and the protein content in tissue homogenate are determined at the
same time. a unit enzyme activity is indicated by the quantity of the
enzyme that produce 1 μmol NADP+ (or NDA+) in one minute.
The enzyme activity of the organizations is showed with specific
activity, the specific activity equal enzyme units/mg protein.

[0087]3.3 Measurements about oxidation

[0088]3.3.1 Preparation of tissue homogenate: Rats were killed by femoral
artery bloodletting after administration of the rats for 12-weeks. Kidney
tissue of the rats were taken, the blood of kidney tissue were washed
with cooling saline (4° C.), then the remnant water was absorbed
with filter paper, the kidney tissue was weighed. Per gram kidney tissue
was added into 10 ml saline, which was put in homogenized machine (10,000
r/min, 10 seconds, 3rd), 10% tissue homogenate was prepared, the tissue
homogenate was centrifuged (4000 r/min) for 10 minutes to get
supernatant, the supernatant was saved in -20° C. for
determination.

[0096]These results suggest that the composition that is composed of
mangiferin and berberine has preferable hypoglycemic effect compared with
mangiferin and berberine which is used alone.

[0097]Because the composition has the hypolipidemic effect and improving
the oxidation measurements of kidney and the AR activity of lens, the
composition has the effect of prevention and treatment of chronic
diabetic complications.

The Effect of the Different Ratio Compositions in GK Rats

1. Material

[0098]1.1 Sample: Mangiferin and berberine are prepared in accordance with
the method of preparation of example 1 and 2. Then they can be mixed in
accordance with the dose and the ratio such as table 3.

[0100]In addition to the normal rats, the other rats were fed with high
fat diet (20% protein and 3.5% cholesterol).

2. Experimental Group:

[0101]After the rats was feeding adaptation for one week, GK rats that the
blood glucose level of the GK rats was more than 11.1 mmol/L were
selected, and then random divided into the following groups as follows
(Table 3):

[0102]There are ten rats in every group. GK rats control group and wistar
rats group were given saline. The other groups were given test drugs.
Test samples were administrated orally at the same time every day for 12
weeks.